Clinical Utility Of ctDNA Research Articles

Current Clinical Utility of Circulating Tumor DNA Testing in Breast Cancer: A Practical Approach.

Circulating tumor DNA (ctDNA) refers to DNA fragments released from cancer cells into the bloodstream. Clinical utility of ctDNA in breast cancer has been explored in both metastatic breast cancer (MBC) and early-stage breast cancer (EBC) settings. In MBC, ctDNA can detect therapeutically targetable genomic alterations and has shown great potential in predicting treatment response or resistance. Accumulating data suggest that ctDNA might also have prognostic value in MBC. In EBC, emerging data have shown ctDNA's predictive and/or prognostic value in both neoadjuvant and adjuvant settings. Minimal residual disease (MRD) detection via ctDNA to detect clinical recurrence after curative therapy is a rapidly advancing field. In this review, we discuss the existing and emerging data regarding ctDNA utility in both MBC and EBC settings.

Clinical utility of ctDNA by amplicon based next generation sequencing in first line non small cell lung cancer patients.

The assessment of ctDNA has emerged as a minimally invasive avenue for molecular diagnosis and real-time tracking of tumor progression in NSCLC. However, the evaluation of ctDNA by amplicon-based NGS has been not endorsed by all the healthcare systems and remains to be fully integrated into clinical routine practice. To compare tissue single-gene with plasma multiplexed testing, we retrospectively evaluated 120 plasma samples from 12 consecutive patients with advanced non-squamous NSCLC who were part of a prospective study enrolling treatment-naïve patients and in which tissue samples were evaluated using a single-gene testing approach. While the plasma ctDNA detection of EGFR and BRAF mutations had an acceptable level of concordance with the archival tissue (85%), discordance was seen in all the patients in whom ALK alterations were only detected in tissue samples. Among six responders and six non-responders, early ctDNA mutant allelic frequency (MAF) reduction seemed to predict radiologic responses and longer survival, whereas increasing MAF values with the emergence of co-mutations like BRAFV600E, KRASG12V or TP53M237I seemed to be an early indicator of molecular and radiologic progression. This report using an amplicon-based NGS assay on ctDNA underscores the real-life need for plasma and tissue genotyping as complementary tools in the diagnostic and therapeutic decision-making process.

Clinical utility of ctDNA as a tool to detect triple-negative breast cancer relapses: The CUPCAKE trial.

TPS1139 Background: The current follow-up of triple-negative breast cancer (TNBC) patients at high risk of relapse does not involve any blood or imaging monitoring to detect metastases, because of the low sensitivity and specificity of blood and imaging biomarkers in that context. In contrast, ctDNA detection techniques are highly specific, sensitive and can detect a molecular relapse several months before the occurrence of symptoms (1). We hypothesize that the early detection of molecular relapse by ctDNA combined with whole-body mapping of tumor recurrence sites by 68Ga-FAPI-46-PET/CT will enable the detection of relapses when they are still limited in size and number of sites. Detecting oligometastatic relapses could demonstrate a clinical utility by (i) allowing the use of local treatments in addition to systemic treatments; (ii) taking advantage of the higher efficacy of immunotherapy in patients with limited tumor burden and inflammation. Methods: CUPCAKE is a randomized, non-comparative, multicenter trial promoted by Institut Curie (Paris, France), which design is inspired from the Trials WIthin CohortS approach (2). N=450 non-metastatic TNBC patients at high risk of relapse will be included after having signed a written informed consent in a cohort allowing them to be followed by ctDNA monitoring. For each included patient, a ctDNA detection assay (FoundationOne Tracker) will be performed every 3 months, with a turnaround time of less than 3 weeks. When negative, ctDNA detection results will not be disclosed to patients nor clinicians. If, at any timepoint, ctDNA is detected (molecular relapse), patients will be randomized in a 1:1 ratio between the experimental arm (ctDNA-based intervention) and the control arm (no intervention). In the experimental arm, the positive ctDNA result will be disclosed. To locate metastatic foci, patients will be offered to undergo whole-body imaging with 18F-FDG and 68Ga-FAPI-46-PET/CT, in addition to any other workup considered as relevant by their treating physician. If/when a clinical/radiological relapse is observed, systemic or local treatments will be decided by physicians (standard of care). In the control arm, patients and their treating physician will not be made aware of the molecular relapse and will continue the standard surveillance with repeated ctDNA test every 3 months (blinded). At the time of the clinical/radiological diagnosis of relapse, similar procedures will be performed (18F-FDG & 68Ga-FAPI-46-PET/CT). The primary endpoint is overall survival rate at 24 months after the randomization in the experimental arm while the control arm will serve as reference. Secondary endpoints include efficacy criteria like progression-free survival but also clinical features upon radiological/radiological relapse, or performance criteria of 68Ga-FAPI-46 versus 18F-FDG PET/CT. 1. Coombes, Clin Cancer Res. 2019. 2. Relton, BMJ. 2010. Clinical trial information: 06225505.

Genomic profiling and clinical utility of circulating tumor DNA in metastatic prostate cancer: SCRUM-Japan MONSTAR SCREEN project

BackgroundCirculating tumor DNA (ctDNA) testing has emerged as a novel tool for cancer precision medicine. This study investigated the genomic profiling and clinical utility of ctDNA in metastatic prostate cancer.MethodsThis is a nation-wide prospective observational study. Patients treated with systemic treatment for metastatic castration-sensitive prostate cancer (mCSPC) and metastatic castration-resistant prostate cancer (mCRPC) were included. ctDNA was analyzed using FoundationOne Liquid®CDx at enrollment. In a subset of patients, ctDNA after disease progression and tissue prior to the initiation of treatment were examined using FoundationOne Liquid®CDx and FoundationOne®CDx, respectively.ResultsThe frequency of AR alterations and homologous recombination repair (HRR) defect was higher in mCRPC compared with mCSPC. Tumor mutational burden was correlated between tissue and ctDNA at pre-treatment, as well as ctDNA between at pre-treatment and at post-treatment. Patients with HRR defect were associated with shorter time to castration resistance in androgen deprivation therapy/combined androgen blockade, but not in androgen receptor pathway inhibitor, compared with patients without HRR defect in mCSPC. Time to treatment failure in patients with AR amplification or AR mutation was shorter compared with patients without AR alterations in mCRPC.ConclusionsThis study revealed valuable findings for the clinical care of metastatic prostate cancer. Especially, predictive factors such as HRR defect in mCSPC should be validated in the future.

Practical recommendations for using ctDNA in clinical decision making.

The continuous improvement in cancer care over the past decade has led to a gradual decrease in cancer-related deaths. This is largely attributed to improved treatment and disease management strategies. Early detection of recurrence using blood-based biomarkers such as circulating tumour DNA (ctDNA) is being increasingly used in clinical practice. Emerging real-world data shows the utility of ctDNA in detecting molecular residual disease and in treatment-response monitoring, helping clinicians to optimize treatment and surveillance strategies. Many studies have indicated ctDNA to be a sensitive and specific biomarker for recurrence. However, most of these studies are largely observational or anecdotal in nature, and peer-reviewed data regarding the use of ctDNA are mainly indication-specific. Here we provide general recommendations on the clinical utility of ctDNA and how to interpret ctDNA analysis in different treatment settings, especially in patients with solid tumours. Specifically, we provide an understanding around the implications, strengths and limitations of this novel biomarker and how to best apply the results in clinical practice.

Is liquid biopsy useful in patients with GI malignancies and exclusive peritoneal disease?

809 Background: Peritoneal carcinomatosis (PC) is a common manifestation in gastro-intestinal (GI) cancers.10-20 % of patients (pts) will present isolated, unresectable peritoneal disease with poor prognosis. In the past years, circulating tumor DNA (ctDNA) has emerged as a novel and less invasive biomarker for molecular diagnosis and disease monitoring. We aimed to evaluate the ability of PC to shed ctDNA into circulation in a cohort of pts with GI cancers and isolated PC treated in our institution. Methods: We reviewed retrospectively the results of liquid biopsies obtained at progression or at diagnosis. Genomic analysis was done using Foundation One liquid CDx panel. Data on demographics, tumor type, clinical outcomes and molecular alterations (MA) have been collected. Results: Out of 400 GI pts tested, 85 with exclusively PC were included in the analysis. The main characteristics were as follows: 45(53%) female, median age of 57 years (IQR: 24-79), majority have received systemic chemotherapy (all were progressive) (n=63, 74%) with median number of previous lines 1 (range: 0-3). Most common tumor types were as follows: gastric (n=24, 28%), colorectal (n=22, 26%) and primary peritoneal (n=20, 24%) cancers. The median index of peritoneal carcinomatosis was 23(IQR: 6-39). The tumor fraction was very low or undetectable in a proportion of 81% (n=69), median Tumor Mutational Burden was 1 mutation per megabase and was not correlated to tumor volume. Gene alterations were found in a proportion of 56% of pts (n=48) but most common alteration were TP 53 (n=22 pts, 26%) and KRAS mutation (n=14 pts, 16%). Targetable MA were identified only in 11 pts (13%) as follows: ATM, ARID1A, BRAFV600E, ERBB2, krasG12C. Three of them were included in a clinical trial. Germline mutations were found in 5 pts: ATM, CDH1, BRCA2, MSH6, BAP1. For colorectal pts known KRAS and BRAF V600E mutations were confirmed in plasma in a proportion of 32% (7 pts out of 22), for pancreatic cancer the KRAS in 17% (3 out of 18) and for gastric cancer, ERBB2 amplification in 5% (1 out of 22 pts). In pts with primary peritoneal disease: pseudomyxoma and mesothelioma, one patient harbored a known germline BAP 1 and kras G12C mutation without other targetable MA. The median survival in MA group was higher compared to non-MA. Conclusions: Isolated Pc and primary peritoneal cancers have distinct clinical and biological characteristics. The clinical utility of ctDNA seems to be limited in isolated PC or primary peritoneal cancers. However, it is not very clear if this to tumor location or to the ability of peritoneum to seed ctDNA.

245. CLINICAL UTILITY OF CTDNA IN LONGITUDINAL MONITORING OF TREATMENT RESPONSES AND RELAPSE IN METASTATIC ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Esophageal squamous cell carcinoma (ESCC) is a deadly cancer frequently diagnosed only at advanced stages, contributing to high mortality. In this study, circulating tumor DNA (ctDNA), which is DNA shed from the tumor into the bloodstream, was utilized for real-time monitoring of treatment response and early detection of relapse using next-generation sequencing. The clinical utility of ctDNA as a biomarker for monitoring disease burden and treatment response was evaluated. A total of 72 metastatic ESCC patients, who received palliative chemotherapy, were recruited. Blood was collected from the patients before, during, and at the end of treatment. ctDNA was extracted from plasma and the mutation profiles were determined by targeted-capture sequencing using the Roche AVENIO ctDNA expanded 77-genes kit. Mutations or amplification detected in the ctDNA were validated by Sanger sequencing or digital PCR. Mutations in the tumors were validated using formalin-fixed paraffin-embedded (FFPE) tissue with the Roche AVENIO Tumor tissue expanded 77-genes kit. Single nucleotide variations (SNVs) were detected in 92% and copy number variations (CNVs) were detected in 22% of the metastatic ESCC patients. The most frequently mutated genes were TP53, NFE2L2 and PIK3CA. The presence of TP53 mutations at pre-cycle III treatment was associated with poor progression-free survival and overall survival. Longitudinal ctDNA analysis showed good concordance between mutant allele frequency (MAF) and tumor burden and the change in MAF preceded the radiographic relapse with an average of 46 days. ctDNA also demonstrated the ability to capture inter-tumor heterogeneity in the primary tumor and lymph node metastasis. Using the 77-gene panel, mutations were detected in ctDNA of metastatic ESCC patients; the change in MAF reflected the tumor burden. Serial analysis of ctDNAs indicates the usefulness of ctDNA as a prognostic and predictive biomarker for real-time non-invasive monitoring to assess tumor burden and to predict treatment efficacy, disease relapse, and patient survival. Mutation profiles in ctDNA may also be useful in guiding personal targeted therapy.

P1.16-02 Clinical Utility of ctDNA in Advanced NSCLC at Diagnosis or Where Insufficient Tissue Was Available, Based on the ESMO ESCAT Scale

Comprehensive genomic profiling (CGP) by next generation sequencing (NGS) of ctDNA can identify a wide spectrum of genomic alterations that range from driver oncogenic alterations with FDA/EMA approved targeted therapies for routine use, to other alterations with lack of evidence for actionability. We aimed to assess the clinical utility of NGS based ctDNA genomic profiling, based on the ESMO scale for clinical actionability of molecular targets (ESCAT), in a large prospective cohort of advanced NSCLC patients.

Abstract 5766: Pan-cancer analysis revealed characteristics of reversion mutations in homologous recombination repair genes

Abstract Background: Reversion mutations of BRCA1/2 are associated with decreased response to platinum-based chemotherapies and PARP inhibitors. While patient with homologous recombination repair (HRR) genes have proven efficacy to PARP inhibitors, reversion mutations in these genes have not been widely studied. Here, we performed pan-cancer analysis to reveal the characteristics of HRR reversion mutations. Methods: We assessed 1,992 patients with at least one loss of function mutation in BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, RAD54L, ERCC3, RAD51, WRN, FANCI, MRE11A, ATR, FANCD2, FANCA, FANCC, BLM and NBN. Somatic reversion mutations were indel or missense mutations that restore the open reading frame and potentially protein function fully or partially. Definitive reversion mutations are confirmed secondary mutations in patients with known pathogenic germline HRR gene mutations, or in a subsequent sample of previously detected somatic pathogenic mutants. Other reversion mutations are putative due to lack of matched samples. Results: Overall, we found 31/1,992 (1.6%) tumors with definitive or putative HRR reversion mutations, including 17 with germline and 14 with somatic predisposed pathogenic HRR mutations. Of these, we identified 9 BRCA1 (29%, 2 sBRCA1 and 7 gBRCA1) and 12 BRCA2 (39%, 4 sBRCA2 and 8 gBRCA2). We also found pathogenic germline PALB2 (1, 3%) and RAD41D (1, 3%), and somatic mutations in CDK12 (3, 9%), ATM (2, 6%), WRN (1, 3%), FANCD2 (1, 3%), and ATR (1, 3%). Overall, 43 reversion mutations were detected (37 definitive and 6 putative), with eight patients carrying multiple reversion mutations. Importantly, 40/43 reversion mutations were found within 2bp upstream to the start site of original deleterious variants, and all were unique to currently known database. Missense-induced reversions were found in 74% primary stop-gain mutations, while indel-induced reversions were associated with 59% primary frameshift mutations. These HRR reversion mutations were primarily found in breast cancer (9%, 10/107) and ovarian cancer (6%, 3/54), and also in cases of prostate (1/25, 4%), pancreatic (1/29, 3%), colorectal (2%, 9/418), and non-small cell lung cancer (1%, 6/703). Of note, the incidence of multiple HRR reversion mutations was higher in breast cancer (75%, 6/8). Lastly, reversion mutations were detectable in plasma, pleural and ascites fluid samples, suggesting great clinical utility of ctDNA to monitor reversion mutations. Conclusions: This study analyzed clinical and mutational characteristics of reversion mutations in HRR genes, which could occur as single or multiple variants to restore the function of predisposed pathogenic HRR mutations, resulting in resistance to platinum-based chemotherapies or PARP inhibitors. Monitoring these mutations with tissue or liquid biopsy samples are crucial for treatment guidance. Citation Format: Jing Li, Xiaoying Wu, Yan Shi, Di Wang, Yedan Chen, Yang Shao. Pan-cancer analysis revealed characteristics of reversion mutations in homologous recombination repair genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5766.

Clinical utility of circulating tumor DNA in patients with head and neck cancers.

e18003 Background: Globally, head and neck cancers (HNCs) account for over 900,000 cases annually with a meager overall survival rate of 50%. While early-stage HNCs may be amenable to surgery, advanced disease is treated with systemic therapy or chemoradiation. For localized disease, chemo-radiation is often curative, but is associated with severe toxicity. Assessment of systemic treatment in the era of immunotherapy is often challenging due to the pseudoprogression phenomenon. In general, surveillance is limited to clinical follow-up and radiological imaging due to the lack of a sensitive and specific biomarker to assess treatment response and disease progression. Circulating tumor DNA (ctDNA) has shown promise as a reliable marker of disease activity in several solid tumors. In this study we evaluated the clinical utility of ctDNA for monitoring of therapeutic response and disease progression in patients with HNCs. Methods: ctDNA analysis was performed utilizing a personalized, tumor-informed ctDNA assay (Signatera™ bespoke mPCR NGS assay) in patients with advanced stage (III, IV) HNC who underwent chemoradiation or systemic therapy. ctDNA levels were measured at diagnosis, throughout treatment and during remission. The primary endpoint was correlation of ctDNA kinetics with radiological response. The secondary endpoint was the rate of ctDNA positivity during remission. Additional data collected included patient demographics and tumor clinicopathologic characteristics. Analysis was conducted using descriptive statistics. Results: ctDNA kinetics from 15 patients with squamous HNCs were analyzed. Due to insufficient time points, 5 five patients were excluded from this analysis. At diagnosis, 9/10 (90%) patients were ctDNA-positive, with baseline ctDNA levels ranging from 0.2 to 8500 mean tumor molecules (MTM)/mL of plasma. Patients received either chemoradiation (7/10) or (chemo)immunotherapy (3/10). Among patients who received chemoradiation, 4 out of 7 cleared ctDNA in less than 40 days, while 2 out of 7 had persistent ctDNA after 40 days. Those who showed ctDNA clearance had a continued response at 3-6 month follow-up both by ctDNA and by standard of care imaging. The 2 patients that did not clear ctDNA by day 40 ultimately relapsed on imaging and had rising ctDNA levels within 3 months of completion of chemoradiation. The patient who was ctDNA negative at baseline remained negative throughout the treatment and follow up. Of the patients who received chemo(immunotherapy), 2/3 cleared ctDNA after 2 cycles of therapy and continued to be in remission. The remaining patient who did not show ctDNA clearance became refractory to treatment. Conclusions: Our study demonstrates the feasibility of personalized and tumor-informed ctDNA in patients with advanced HNCs. Monitoring of ctDNA kinetics during therapy may enable prediction of therapeutic response and early disease progression to guide clinical decision-making.

A protocol to clinically evaluate liquid biopsies as a tool to speed up diagnosis of children and young adults with aggressive infection-related lymphoma in East Africa \u201c(AI-REAL)\u201d

BackgroundThe capacity for invasive tissue biopsies followed by histopathology diagnosis in sub-Saharan Africa is severely limited. Consequently, many cancer patients are diagnosed late and outcomes are poor. Here, we propose to evaluate circulating tumour (ct) DNA analysis (“liquid biopsy”), a less invasive and faster approach to diagnose endemic EBV-driven lymphomas (EBVL) in East Africa.MethodsWe will evaluate the clinical utility of an already validated ctDNA test prospectively in a head-to-head comparison against histopathology. The primary endpoint is the time from presentation to the specialist centre to a final diagnosis of EBV- Lymphoma. Secondary endpoints include the sensitivity and specificity of liquid biopsy and health economic benefits over histopathology. One hundred forty-six patients will be recruited over 18 months. Patients will be eligible if they are 3–30 years of age and have provided written consent or assent as per IRB guidelines. Tissue and venous blood samples will be processed as per established protocols. Clinical data will be captured securely and in real-time into a REDCap database. The time from presentation to diagnosis will be documented. The sensitivity and specificity of the methods can be estimated within 5% error margin with 95% confidence level using 73 cases and 73 controls. Health-economic assessment will include micro-costing of ctDNA test and histopathology. All results will be reviewed in a multidisciplinary tumour board.DiscussionThe study evaluates the clinical utility of ctDNA in improving the speed of diagnostic pathways for EBVL in sub-Saharan Africa. Our results would provide proof-of-principle that ctDNA can be used as a diagnostic tool in areas without access to regular pathology, that transfer of the tool is feasible, and that it leads to an earlier and faster diagnosis. The potential clinical and economic impact of this proposal is thus significant. If successful, this study will provide appropriate, and cost-effective diagnostic tools that will promote earlier diagnosis of EBVL and potentially other cancers in countries with restricted healthcare resources.Trial registrationPan African Clinical Trials Registry: PACTR202204822312651, registered on 14th-April-2022.

617P Genomic profiling of circulating tumor DNA in advanced genitourinary cancer patients: SCRUM-Japan MONSTAR SCREEN Nationwide Cancer Genome Screening Project

Circulating tumor DNA (ctDNA) is an emerging resource for the diagnosis and prognosis of various types of cancer. However, characteristics and clinical utility of ctDNA is still largely unknown, especially in patients with genitourinary (GU) cancers.

Accurate early-stage colorectal cancer detection through analysis of cell-free circulating tumor DNA (ctDNA) methylation patterns.

3606 Background: Colorectal cancer (CRC) screening programs suffer from poor uptake and biomarkers have limited diagnostic accuracy. The measurement of the methylation status of tumor-derived cell-free DNA in plasma may address these challenges. We used a targeted methylation panel, tumor-derived signal deduction and machine learning algorithm to refine a blood test for the detection of early-stage CRC. Methods: This was a prospective, international multicenter observational cohort study. Plasma samples were collected either prior to a scheduled colonoscopy as part of standard colorectal cancer screening or prior to colonic surgery for primary CRC. Differentially methylated regions (DMRs) were initially selected by analyzing CRC and control tissue samples with whole genome bisulfite sequencing. A targeted sequencing assay was designed to capture these DMRs in plasma ctDNA. Individual sequencing reads were evaluated for cancer-specific methylation signal and scores calculated for each DMR in a sample. A panel of methylation scores originating from 203 DMRs was used in a prediction model building and validated in a test cohort of patients. Results: Calculated scores were used to train a machine learning model on 68 ctDNA samples from 18 early stage (I-II) and 16 late-stage (III-IV) CRC patients and 34 age, BMI, gender and country of origin matched neoplasia-free controls (median age 63 [50-74], mean BMI 27 [19.5-37], female 50%, Spanish and Ukrainian population, distal cancers 50%). This model was then applied to an independent set of subjects from Spanish, Ukraine and Germany, including 36 stage I-IV cancer patients (median age 61.5 [55-82], BMI 28 [16-39], female 47%, 42% of the tumors were distal) and 159 age and sex matched controls. 87 of the control patients had a negative colonoscopy finding (cNEG), 19 had hyperplastic polyps (HP), 37 had small non-advanced adenomas (NAA) and 16 were diagnosed with other benign gastrointestinal diseases (GID). The model correctly classified 92% (33/36) of CRC patients. Sensitivity per cancer stage ranged from 83% (5/6) for stage I, 92% (11/12) for stage II, 92% (12/13) for stage III to 100% (5/5) for stage IV. Specificity of the model was 97% (154/159), with 100% (37/37) NAA, 94% (15/16) GID, 95% (18/19) HP and 97% cNEG patients correctly identified. Lesion location, gender, BMI, age and country of origin were not significantly correlated to prediction outcome. Conclusions: Methylation sequencing data analyzed using read-wise scoring approach combined with a machine-learning algorithm is highly diagnostic for early-stage (I-II) CRCs (89% sensitivity at 97% specificity). This method could serve as the basis for a highly accurate and minimally invasive blood-based CRC screening test with significant implications for the clinical utility of ctDNA in early-stage cancer detection.

Landscape of genomic alterations of circulating tumor DNA in advanced genitourinary cancer patients: SCRUM-Japan MONSTAR SCREEN Project.

152 Background: Circulating tumor DNA in plasma (ctDNA) is an emerging resource for detecting genomic alterations in various cancers. However, the characteristics and clinical utility of ctDNA are not fully elucidated, especially in patients with genitourinary (GU) cancers. Methods: In April 2019, SCRUM-Japan started the MONSTAR-SCREEN project, which evaluates the ctDNA from patients with various advanced solid tumors. We collected plasma and tumor samples from patients with advanced prostate cancer (PC), urothelial carcinoma (UC), and renal cell carcinoma (RCC). Plasma ctDNA and tissue genomic DNA were analyzed using NGS-based cell-free DNA assay, a modified version of FoundationOne Liquid (F1L) including blood tumor mutational burden (TMB) analysis, and tissue-based panel, FoundationOne CDx (F1CDx), respectively. Success rate of ctDNA was defined as the percentage of patients whose sample quality control status was “pass” or “qualified”. Level of ctDNA was defined as the maximum allele fraction (AF) for all known somatic alterations detected. Results: As of June 18, 2020, ctDNA analysis results were available for 470 of 540 patients with advanced solid tumors. Among them, we analyzed 70 advanced GU cancers (35 PC, 17 UC, and 18 RCC) and 400 non-GU cancers. The success rate of ctDNA was significantly lower in GU cancers than in non-GU cancers (81.4% vs. 91.5%, P = 0.016). The levels of ctDNA in PC and UC were similar to those in non-GU cancers. RCC had the second lowest ctDNA level (median 0.13%) after that in malignant melanoma. The median TMB, as estimated by ctDNA, was 4.40, 0.88 and 0.44 mutations/Mb in UC, PC and RCC, respectively. The most frequently altered genes were TP53 (34%), AR (11%), BRCA2 (11%), ATM (8.6%), and CDK12 (8.6%) in PC, TP53 (59%), TERT (41%), and CHEK2 (18%) in UC, and TP53 (22%), ATM (11%), and MTOR (11%) in RCC. The mutation rate in genes related to DNA damage response (DDR) pathways was significantly higher in GU cancers compared to that in non-GU cancers (30.0% vs. 18.0%, P = 0.033). However, other pathways were less frequently altered in GU cancers versus non-GU cancers, including Wnt (5.7% vs. 16.8%, P = 0.017), PI3K (8.6% vs. 19.0%, P = 0.039) and RAS/MAPK (8.6% vs. 29.5%, P < 0.001). Conclusions: Our results reveal the genomic landscape of ctDNA in several advanced solid tumors, and highlight the differences between tumor types. Comprehensive analysis of ctDNA using the F1L assay reveled alterations in DDR-associated genes were significantly more frequent in GU cancers than in non-GU cancers. Clinical trial information: UMIN000036749.

Abstract 708: Limited release of circulating tumor DNA into the systemic circulation by peritoneal metastases from colorectal cancer

Abstract Introduction: Circulating tumor DNA (ctDNA) from plasma is a promising biomarker. Patients with metastatic colorectal cancer (CRC) often have high ctDNA levels compared to other cancer types. However, it is unclear whether this is also the case for patients with peritoneal metastases from CRC. Aim: To compare the propensity of ctDNA shedding by isolated liver metastases and isolated peritoneal metastases from CRC. Methods: Plasma was collected from 100 CRC patients (64% male, mean age of 60 [SD=10] years) with isolated unresectable liver metastasis (CRC-LM). Plasma and ascites were obtained from 20 CRC patients (60% male, mean age of 63 [SD=9.8] years) with isolated unresectable peritoneal metastases (CRC-PM). All liquid biopsies were obtained prior to treatment and cell-free DNA was isolated using the QIAsymphony (Qiagen, Germany) and assessed by droplet digital PCR (ddPCR; Bio-Rad, Hercules, USA). Patients with a KRAS or BRAF tumor tissue mutation were suited for plasma ctDNA ddPCR analysis, and were detected in 57 CRC-LM (57%) and 11 CRC-PM (55%) patients. The ability to shed ctDNA into the circulation was evaluated by comparing KRAS/BRAF mutation status in plasma and ascites with the mutation status based on tumor tissue. Regarding liquid biopsies, mutant allele fraction (MAF) and mutant copies per ml input (MTc/ml) were reported. Results: Tissue mutations could be confirmed in plasma in 93% of CRC-LM and only 20% of CRC-PM patients, whereas mutations were detected in ascites in 100% of CRC-PM patients. The MAF and MTc/ml were both significantly lower in CRC-PM plasma ctDNA (median MAF=0.28% and MTc/ml=21) compared to CRC-LM plasma ctDNA (median MAF=18.9% and MTc/ml= 1758; P<0.0001). In addition, the MAF and MTc/ml were significantly higher in CRC-PM ascites ctDNA (median MAF=16.4%, MTc/ml=305) compared to plasma ctDNA (median MAF=0.28%, MTc/ml=21; P<0.0001). Conclusion: To our knowledge, this is the first study showing a comprehensive comparison of tissue, blood and ascites derived genomic information in patients with CRC and extensive isolated peritoneal metastases. This study concludes that isolated peritoneal metastases from CRC is a malignancy with distinct clinical and biological characteristics, which should be taken into account when considering the clinical utility of ctDNA in different metastatic setting of CRC. To detect genomic alterations, the blood circulation is the preferred source of ctDNA in case of CRC liver metastases, whereas ascites offers an alternative to plasma in patients with peritoneal metastases, which might be suitable as diagnostic, prognostic, predictive or disease monitoring biomarker. Citation Format: Iris van 't Erve, Koen P. Rovers, Alexander Constantinides, Karen Bolhuis, Emma C. Wassenaar, Robin J. Lurvink, Clément J. Huysentruyt, Petur Snaebjornsson, Daan van den Broek, Tineke E. Buffart, Niels F. Kok, Gerrit A. Meijer, Cornelis J. Punt, Onno Kranenburg, Ignace H. de Hingh, Remond J. Fijneman. Limited release of circulating tumor DNA into the systemic circulation by peritoneal metastases from colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 708.

Abstract A43: Plasma-derived circulating tumor DNA (ctDNA) as a surrogate biomarker for treatment response with the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with LDAC or decitabine in acute myeloid leukemia (AML)

Abstract Background: Cancer cells from solid tumors release ctDNA into the bloodstream, clinically enabling assessment, monitoring tumor mutational heterogeneity, treatment response, minimal residual disease (MRD), and disease progression. However, the clinical utility of ctDNA in AML has yet to be established. In AML, blast cells carrying a driver mutation originate in the bone marrow (BM) and migrate into circulation in peripheral blood (PB). Here, we aimed to determine the potential of ctDNA as a surrogate for response to treatment in the phase 1b/2 study (NCT03303339) testing the efficacy and safety of the PLK1 inhibitor, onvansertib, in combination with either decitabine or LDAC in relapsed or refractory AML (R/R AML). Methods: R/R AML patients were dosed for 5 days with onvansertib in combination with either decitabine (5 days) or LDAC (10 days) within 21 to 28-day cycle. BM aspirates and blood samples were collected pre-dose, at the end of cycles 1, 2 and every other cycle after. Genomic DNA (gDNA) was extracted from BM and PB mononuclear cells (BMMCs and PBMCs, respectively), while ctDNA was extracted from plasma. Clinically significant mutations were detected in gDNA from PBMCs and BMMCs using a next-generation sequencing panel targeting variants in 75 genes associated with AML. For each patient, a droplet digital PCR (ddPCR) assay was developed to assess mutant allele frequencies (MAF) of a single variant of interest across timepoints and three sample sources (BMMC, PBMC, and Plasma). MAF changes across all 3 sample sources were compared to the clinical response assessed by immunohistochemistry in bone marrow aspirate (BM-IHC). Results: As of October 16th, 16 patients were evaluated, totaling 33 timepoints with MAF measured in all 3 sources. MAF in plasma highly correlated with BMMCs MAF (R2 = 0.85), more so than PBMCs with BMMCs (R2 = 0.78) and over plasma with PBMCs (R2 = 0.68). Clinically, 4 patients had a complete response (with or without count recovery, CR+CRi) and showed a corresponding drop in ctDNA MAF at or before clinical diagnosis. Similarly, 3 of 4 evaluable patients with progressive disease showed an increase in ctDNA MAF before or at the timepoint when blast percentages rose in BM, with the fourth patient maintaining a stable MAF across timepoints. In 5 patients with stable disease, 2 showed a divergence between ctDNA MAF and BM-IHC, while 3 maintained congruency. Conclusions: Serial testing of plasma-derived ctDNA was representative of treatment response and relapse in R/R AML patients. The correlation between ctDNA MAF and BMMCs MAF supports the utility of ctDNA as a surrogate biomarker for disease status, and in some cases provides a more immediate diagnostic of response or nonresponse. More patients need to be evaluated to build an objective classification of clinical significance around ctDNA MAF, and, importantly, the biology of R/R AML subclonal heterogeneity and MRD likely necessitate interrogation and monitoring of multiple variants of interest. Citation Format: Errin Samuelsz, Maya Ridinger, Mark Erlander, Latifa Hassaine, Marion Luebbermann, Brittany Ross. Plasma-derived circulating tumor DNA (ctDNA) as a surrogate biomarker for treatment response with the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with LDAC or decitabine in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A43.

Abstract B10: Feasibility of bronchial washing fluid-based approach to early-stage lung cancer diagnosis

Abstract Circulating tumor DNA has few applications for detecting early-stage lung cancers. Bronchial washing (BW) is minimally invasive, and BW fluids can contain tumor DNA. Thus, analysis of BW fluids could be used for precision medicine. Forty-eight paired specimens (primary tumor tissue, normal tissue, BW supernatant, and BW precipitate) from twelve patients with early-stage non-small cell lung cancer were analyzed using ultradeep next-generation sequencing with a custom panel containing 113 genes. In primary tumors, 130 missense mutations/indels (5–16 per patient) and 20 driver mutations (0–3) were found; 105 (80.8%) and 97 (74.6%) of these missense mutations/indels were identified in BW supernatants and precipitates, respectively, and 19 (95.0%) of the driver mutations in both BW supernatants and precipitates. Concordance between EGFR mutations in primary tumors, BW supernatants, and BW precipitates was 100%. The median allele frequencies (AFs) for missense mutations/indels in primary tumors, BW supernatants, and BW precipitates were 13.0%, 0.3%, and 0.1%, respectively. The AFs of missense mutations/indels in BW supernatants correlated with those in primary tumors and in BW precipitates (P<0.001). In conclusion, this study provides evidence that the BW fluids, especially supernatants, can be an alternative to overcome the limitation that clinical utility of ctDNA is often not reliable in early-stage NSCLC. Citation Format: Jeong Seon Ryu, Myoung Kyu Lee, Seung Jae Lee. Feasibility of bronchial washing fluid-based approach to early-stage lung cancer diagnosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B10.

Abstract P5-01-08: Landscape of circulating tumor DNA (ctDNA) in metastatic breast cancer

Abstract Background: The clinical utility of routine clinical assessment of ctDNA in metastatic breast cancer (MBC) has not been clearly established. Recently, the Food and Drug Administration approved alpelisib (Piqray®) with a ctDNA-based companion diagnostic. Therefore, there is significant interest in describing the landscape of ctDNA alterations and establishing further clinical utility of this assessment. Methods: We retrospectively identified ctDNA data from a single institution (Northwestern University). All patients had next-generation sequencing performed by Guardant Health (Redwood City, CA) from 2015-2019 for clinical evaluation, and patients were consented for participation in a prospective registry study. Reports were evaluated for type of genomic alterations, number of alterations, and mutant allele frequency (MAF) of the dominant clone. Clinical information including demographics, pathology, imaging, treatment, and response data were collected via patient chart review. Associations were tested through Fisher’s exact test or Kruskal-Wallis test as statistically appropriate. Matched paired samples were investigated through McNemar test. Results: The cohort consisted of 255 patients with MBC. In total, 371 ctDNA reports were reviewed, including 116 patients with serial assessments. Baseline ctDNA evaluation occurred at a median of 3 lines of therapy (interquartile range (IQR): 1 - 5). Median number of alterations was 4 (IQR: 2-7), while median MAF was 4% (IQR: 0.6% - 17.9%). Across the entire cohort, PIK3CA and TP53 were the most commonly detected mutations, while MYC, FGFR1, PIK3CA, and ERBB2 were the most commonly detected amplifications. 40% of patients had PIK3CA aberrations observed in the analyzed plasma samples (28% amplification, 22% H1047R, 11% E542K), which was independent of breast cancer subtype. In the subset of patients with hormone-receptor positive (HR+) breast cancer, 30% of patients had ESR1 mutations (27% D538G, 19% Y537S, 12% Y537N, 11% E380Q). Patients with triple negative breast cancer (TNBC) had statistically higher MAF compared to HR+ or HER2+ breast cancer (P<0.05). However, there were no differences in the number of alterations observed in each subtype. When examining the mutational profile of subtypes across lines of therapy, only ESR1 and FGFR1 were associated with collection beyond the first line of therapy for HR+ patients (P=0.003 and P<0.025, respectively). The mutational profiles of TNBC and HER2+ subtypes did not vary by lines of therapy at baseline blood collection. Number of genomic alterations detected increased incrementally with increasing lines of therapy (P<0.01), with specific genes more likely to be altered beyond the first line (ESR1, FGFR1, NF1). Higher MAF and number of genomic alterations were also significantly associated with number of metastatic sites on imaging (both P<0.001). In the subset of patients with serial blood collections, across all patients, the following genomic alterations were significantly more likely to emerge over time in paired samples: APC, BRAF, CCNE1, ERBB2, MET, MYC, NF1 (all P<0.05 with Q values corrected for multiplicity). Conclusion: Liquid biopsy evaluation using ctDNA identified clinically meaningful genomic alterations at high frequency. In addition, our data indicate the potential clinical utility of ctDNA as a surrogate for tumor burden on imaging based on the observed correlation with lines of therapy received and sites of disease. Serial assessment of ctDNA demonstrated clonal evolution, particularly in the HR+ cohort, and identified resistant clones. Validation with an independent cohort is warranted. Citation Format: Andrew A Davis, Saya Jacob, Lorenzo Gerratana, Ami N Shah, Firas Wehbe, Neelima Katam, Qiang Zhang, Lisa Flaum, Kalliopi Siziopikou, Leonidas C Platanias, William J Gradishar, Amir Behdad, Massimo Cristofanilli. Landscape of circulating tumor DNA (ctDNA) in metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-08.

Abstract 1353: The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis

Abstract Background : Circulating tumor DNA (ctDNA) is considered to be a new class of tumor-specific personalized biomarkers. Since the variant allele frequency (VAF) of ctDNA is very low (0.01-1%), it is more important to quantify tumor-specific mutations using specific probes than to perform exploratory analyses. A molecular targeting drug is often selected by mutations, and it has not been verified whether the mutations reflect the level of proteins as the drug target molecule. Purpose: To clarify: (a) the utility of ctDNA from tumors with genetic heterogeneity; (b) the ability of early detection for relapse; and (c) the validity of protein level prediction by gene mutation. Methods: Tumor DNA was extracted from three regions of 14 Stage III/IV surgically-resected colorectal tumors. Plasma DNA was also extracted from pre-/post-operative as well as follow-up blood samples every three months. The multi-regional sequencing analysis was conducted using a next generation sequencer (NGS) with a panel of 151 genes and phylogenetic trees were subsequently generated. Of the detected non-synonymous mutations, ctDNA was monitored using digital PCR (dPCR) for which primers and probes were designed by the Hypercool Primer & Probe™ technology to facilitate a specific PCR with short (~70bp) DNA fragments. A reverse phase protein array (RPPA) was used to quantitate 294 proteins in cancer tissues for three regions. Results: The average number of mutations per tumor and per region was nine and six, respectively. The average number of founder mutations (i.e., mutations found in all three regions) and non-founder mutations was three and seven, respectively. Phylogenetic analysis revealed that mutations with high VAF tended to emerge at the trunk of the evolutional tree. Interestingly, when non-founder mutations in the regions that had not exhibited the mutation by NGS were detected using dPCR in 5 of 13 regions. mutations were still detected in 5 of 13 regions (38.5%). The VAFs of founder mutations were significantly higher than those of non-founder mutations. In pre-operative plasma, ctDNA was detected in 10 of 12 patients (83.3%) with low VAF (range; 0.028-13.3%). Founder mutations in ctDNA were detected in 8 of 10 patients with low VAF (range; 0.04-13.3%). In each patient, founder mutations appeared to exhibit higher VAFs in ctDNA than those of non-founder mutations. Importantly, the VAF of ctDNA started to increase 6 months before the relapse was diagnosed by CT scan. Among 42 gene-protein matched pairs, the level of phosphorylated proteins that could be molecular targets did not seem to be predicted by the coding gene mutation. Conclusions: To select mutations for ctDNA monitoring, founder mutations with high VAF in a tumor are mandatory. Since mutations in colorectal cancer do not seem to predict the protein level, the proper clinical utility of ctDNA is a tumor marker over the entire course of colorectal cancer therapy. Citation Format: Mizunori Yaegashi, Takeshi Iwaya, Masashi Fujita, Zhenlin Ju, Doris Siwak, Kei Sato, Fumitaka Endo, Ryo Sugimoto, Tamotsu Sugai, Lance Liotta, Yiling Lu, Gordon Mills, Hidewaki Nakagawa, Satoshi Nishizuka. The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1353.

Identification of genomic alterations by circulating tumor DNA in leiomyosarcoma: A molecular analysis of 73 patients.

11044 Background: Leiomyosarcoma is a malignant mesenchymal tumor of cells of smooth muscle lineage arising commonly in retroperitoneum, uterus, large veins, and the limbs. In contrast to many sarcomas, the genetics of leiomyosarcomas are complex and there is very limited understanding of common driver mutations. Circulating Tumor DNA (ctDNA) offers a rapid and non-invasive method of next-generation sequencing (NGS) that could be utilized for diagnosis, therapy and detection of recurrence. Methods: ctDNA testing was performed using Guardant360, which detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion mutations in 73 genes utilizing NGS. Blood samples from patients with leiomyosarcoma were analyzed, and results from December 2014-December 2018 were reviewed. Results: Of the 90 samples collected and analyzed, alterations were detected in 73 ctDNA samples. After exclusion of variants of uncertain significance (VUS), 63 ctDNA samples harbored cancer-associated genomic alterations. Of 73 patients, 59 were found to have one or more cancer-associated genomic alteration. 76% (45) were female with a median age of 63 (range, 38-87) years. All samples were designated metastatic. The following table shows common alterations and types of mutations. The other alterations included RAF1, ERBB2, MET, PTEN, TERT, APC and NOTCH1. In 24 of the 73 patients (33%) the genomic alterations detected by ctDNA are potentially targetable by an FDA-approved or clinical trial therapy. There were 4 (5%) patients who were found to have incidental germline TP53 mutations. Conclusions: NGS of ctDNA allows identification of genomic alterations in plasma from patients with leiomyosarcoma. Unfortunately, there is limited activity of targeted agents in leiomyosarcomas. These results suggest opportunities to develop therapy against TP53, cell cycle, and kinase signaling pathways. Further validation and prospective evaluation is warranted to investigate the clinical utility of ctDNA for patients with leiomyosarcoma.[Table: see text]